Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 – R = –0.59, YWHAB – R = –0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 – R = –0.46, YWHAB – R = –0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.


Introduction
In humans, reproductive losses are more common in the first trimester of pregnancy than in other periods of embryogenesis. One of the most common causes of early embryonic death is an abnormal number of chromosomes (aneuploidy), which leads to severe developmental anomalies. The formation of aneuploidy with meiotic and mitotic origin corresponds to the waves of epigenetic reprogramming, in particular, genome demethylation in the zygote and at the cleavage stage. Early blastocyst demonstrates less DNA methylation at the latter stage than cells at any other moment of ontogeny (Smith et al., 2012). A rapid wave of de novo DNA methylation for the inner cell mass then follows while the trophectoderm remains hypomethylated (Santos et al., 2010).
Throughout pregnancy, the placenta has a unique hypome thylated epigenetic landscape compared to other extraembryo nic and embryonic tissues, which may indicate its special functions (Robinson, Price, 2015). Hypomethylation in pla cental DNA occurs mainly in "partially methylated domains" and is unevenly distributed throughout the genome. "Partially methylated domains" refers to large (> 100 kb) regions of low DNA methylation alternating with regions of higher DNA methylation (Schroeder et al., 2013).
The placenta exhibits reduced DNA methylation of some types of repetitive genome elements (Price et al., 2012). One of them, the LINE1 retrotransposon (long interspersed nuclear element1), is the largest, occupying approximately 20 % of the genome, and the most evolutionarily young class of retrotransposons in humans, retaining the ability to transpose (Ostertag et al., 2001). The transcriptional activity of LINE1 is suppressed by DNA methylation during most periods of ontogeny.
An important feature of LINE1 that requires attention is its high level of methylation in blood leukocytes, regardless of age and gender, while the level of LINE1 methylation in other tissues has its tissue-specific differences (Chalitchagorn et al., 2004). It was shown that for the placenta as an independent organ, the level of methylation of retrotransposons doesn't always coincide with the global level of methylation of the entire genome. The level of LINE1 methylation in the tissues of the placenta of the third trimester of pregnancy significantly decreases compared to the first trimester of pregnancy. At the same time, changes in the DNA methylation level of the entire genome are not found between the first and third trimester placentas (He et al., 2014).
It can be assumed that LINE1 methylation and activation are transiently regulated during normal placental develop ment. This raises the question of a possible functional role for LINE1 retrotransposon sequences in placental development. Previously, we found that some spontaneous abortions with normal karyotypes were characterized by epigenetic disorders similar to spontaneous abortions with aneuploidy. In particular, some spontaneous abortions with a normal karyotype had an increased methylation index in the LINE1 retrotransposon promoter, which was characteristic of groups of spontaneous abortions with trisomy 16 and monosomy X (Vasilyev et al., 2021b).
One of the possible roles of LINE1 may be the usage of its regulatory sequences to influence the transcription of adjacent genes. This effect becomes feasible because LINE-1 includes 65 ЭПИГЕНЕТИКА И РЕГУЛЯЦИЯ АКТИВНОСТИ ГЕНОВ / EPIGENETICS AND GENE REGULATION Yellow shows proteins that have functional bonds (highlighted in red) with the NUP153 and YWHAB genes (marked in orange) (STRING score > 0.4). a sense promoter that controls the transcription of the ORF1 and ORF2p proteins required for retrotransposition, and an antisense promoter that controls the transcription of chimeric transcripts, LINE-1 5′-antisense sequences spliced with exons of neighboring genes (Denli et al., 2015). LINE1 antisense transcripts affect up to 4 % of all human genes, and LINE-1 antisense promoters are actively transcribed in various types of human cells, including embryonic tissues. A total of 988 genes that can be expressed from alternative LINE1 promo ters have been identified so far (Criscione et al., 2016b). It is possible that the expression of multiple genes in extraembryo nic tissues may occur predominantly from alternative LINE1 promoters because LINE1 promoters are hypomethylated in the placenta.
Using the STRING tool, two genes, NUP153 and YWHAB, were selected among the genes capable of expression from alternative LINE1 promoters. Their products showed a high level of expression in the placenta and are functionally associ ated with proteins involved in trophoblast differentiation (ac cording to Gene Ontology: GO:0061450, trophoblast cell mi gration; GO:0097360, chorionic trophoblast cell proliferation; GO:0001890, placenta development) (Fig. 1). The NUP153 gene functions as a scaffolding element in the nuclear phase of the nuclear pore complex. It is required for normal nuclear cytoplasmic transport of proteins and mRNA during somatic cell division (Bilir et al., 2019) and in mouse embryonic stem cells (Souquet et al., 2018). The YWHAB gene belongs to the group of genes responsible for signal transduction by binding Вавиловский журнал генетики и селекции / Vavilov Journal of Genetics and Breeding • 2023 • 27 • 1 Expression of NUP153 and YWHAB genes from different promoters in human placenta to phosphoserinecontaining proteins. The protein encoded by the gene interacts with RAF1 and CDC25 phosphatases and may play a role in mitogenic signaling and cell cycle regula tory mechanisms. It was shown that YWHAB overexpression stimulates and maintains attachmentindependent cell growth in a fibroblast cell line isolated from mouse embryos (Sasaki et al., 2014).
The aim of this study was to analyze the expression of the NUP153 and YWHAB genes from canonical and alternative LINE1 promoters in the germinal part of the placenta of spontaneous and induced abortions.

Materials and methods
The samples were from chorionic villi and extraembryonic mesoderm of induced abortions (IA) (n = 10, gestational age 8.2 ± 2.3 weeks), spontaneous abortions (SA) with normal karyotype (n = 10, gestational age 7.2 ± 1.4 weeks), trisomy 16 (n = 8, gestational age 6.5 ± 0.8 weeks), and monosomy X (n = 6, gestational age 8.6 ± 0.7 weeks). Samples were taken from the Biobank of Northern Eurasia of the Research Institute of Medical Genetics of the Tomsk National Research Medical Center. The samples were obtained from 2004 to 2021 and stored in liquid nitrogen, before their use for analysis. The study was conducted in compliance with ethical standards by the Helsinki Declaration of the World Medical Association. The study was approved by the Biomedical Ethics Committee of the Research Institute of Medical Genetics of the Tomsk National Research Medical Center (November 9, 2020/No. 7).
A standard cytogenetic analysis was performed on direct preparations of chorionic villi and fibroblast cultures of the extraembryonic mesoderm to determine the karyotype (Le be dev et al., 2004). Karyotyping results for 14 trisomic and mo nosomic SA samples were confirmed by fluorescence in situ hybridization (FISH). Aneuploidy mosaicism was assessed with a lower cutoff of 10 % and an upper cutoff of 90 %.
Centromere-specific DNA probes for X chromosomes were used for the analysis of monosomy X and subtelomeric DNA probes (16q and 16p) were used for the analysis of tri so my 16. The analysis was carried out according to the protocol described elsewhere (Vasilyev et al., 2010). Four samples had a mosaic karyotype with a trisomy level from 10 to 90 %. The remaining 10 spontaneous abortions with a higher propor tion of trisomy or monosomy were classified as having pure aneuploidy. The blood lymphocytes of IA parents (5 couples, age 30.8 ± 2.7) were used as a comparison group that were contained in the Lyra reagent (Biolabmix, Russia) before the start of the experiment.
RNA was isolated from chorionic villi and extraembryonic mesoderm by the phenolchloroform method. All tissues were stored in liquid nitrogen from the moment of obtaining the ma terial of the studied samples to the beginning of RNA isolation. Tissue separation was preliminarily carried out in RNAlater (Invitrogen, USA) to stabilize the RNA in the samples. Each sample was homogenized in a mortar with liquid nitrogen, adding 500 µl of Lyra reagent (Biolabmix, Russia). The lysate was incubated first for 5 min at 55 °C, then for 5 min at room temperature. The lysate was then centrifuged at 12,000 rpm for 10 min to remove undissolved fragments, and the super natant was transferred into a new tube. A volume of 0.2 ml of chloroform was added per each 1 ml of Lyra reagent, followed by shaking (by hand) for 15 s, followed by incubation of the mixture for 10 min at room temperature, and centrifugation at 10,000 g for 10 min at 4 °C. Next, 0.5 ml of 100 % cold iso propanol was added to the aqueous phase containing RNA per each 1 ml of Lyra reagent, and the mix ture was incubated at -20 °C for 10 min, after which the sample was centrifuged at 12,000 g for 10 min at 4 °C.
The precipitate was washed twice with 80 % cold ethanol at 10,000 g for 5 min at 4 °C. The precipitate was then dried for 2 min in a concentrator (Eppendorf, USA) (parameters: 45 °C, V-AL). After this, 40 µl of DEPC water and 1 µl of RiboLock (Thermo, USA) were added to dissolve the precipitate and left for 10 min at room temperature until complete dissolu tion. All samples were kept on ice to avoid RNA degradation during isolation whereas at the incubation stage all steps were performed at room temperature. All samples were stored at -80 °C after isolation.
The RNA was treated with DNase (Biolabmix) to obtain pure RNA. Further, the OTMMuLVRH kit (Biolabmix) with a random hexaprimer was used for reverse transcription. The reverse transcription reaction mixture included 1.5 µg RNA, 3 µl hexaprimer, 4 µl KCl reaction buffer, 2 µl 0.1 M DDT, 1 µl 10 mM dNTP mix, and 1 µl revertase. Two types of primers were designed for the NUP153 and YWHAB genes: the first for long products that are expressed only from ca nonical promoters, and the second for short products that are expressed from alternative LINE1 antisense promoters (see the Table).
The NUP153 gene includes 22 exons, while the short transcript from the alternative LINE1 promoter contains only exons 21-22. Primers were designed in exons 16-17 for detecting NUP153 gene transcripts from the canonical promoter and in exons 21-22 for detecting transcripts from the alternative promoter. Two normal long transcripts with exons 7 or 6 are transcribed from the normal promoter of the YWHAB gene. In this regard, primers were designed for each product. For the first transcript with seven exons, primers were designed in exons 1-2. For the second transcript with 6 exons, primers were designed in exons 1-3. Primers of the short product from the alternative LINE1 promoter of the YWHAB gene were designed in exons 4-7 (Fig. 2). The expression from alternative gene promoters was taken to be the difference between the level of gene expression estimated using primers specific to the region downstream the alternative promoter and the level of gene expression estimated using primers annealing upstream in the first exons. This value was used for data analysis and is displayed on the charts. For the YWHAB gene, the sum of expression levels of both long transcripts was subtracted from the expression level of the canonical promoter.
The methylation index was assessed in 19 CpG sites of the LINE1 promoter in chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. Library preparation and evaluation were carried out according to a previously published protocol (Vasilyev et al., 2021a). Sta tistical analysis of data was performed using Statistica 10.0 software.  The arrows schematically mark the hybridization sites of oligonucleotide primers. The arrow starting at the beginning of the LINE1 element indicates the direction of transcription from the direct LINE1 promoter, which is canonical. The second arrow pointing in the opposite direction marks the direction of expression from the alternative antisense LINE1 promoter, which is also alternative for the studied genes.

Results
The expression level of the NUP153 gene from the canoni cal promoter was 12.5 times higher in lymphocytes than in placental tissues ( p = 0.000001). The expression level of the YWHAB gene from the canonical promoter was also on aver age higher in blood lymphocytes than in placental tissues (by 4.6 times) (transcript NM_13932 ( p = 0.00003)). The expres sion level of the NM_003404 transcript of the YWHAB gene was highly variable in lymphocytes. However, the expression level of the NUP153 gene from alternative LINE1 promoters was statistically significantly higher in extraembryonic tissues compared to lymphocytes of adults (17 times in chorionic villi and 23 times in extraembryonic mesoderm, p < 0.05) (Fig. 3). The levels of expression of both genes from canonical promo ters were higher in the SA group with monosomy X than in the groups of SA with normal karyotype (Fig. 4).

Discussion
In the present work, it was found that the level of expres sion of the NUP153 and YWHAB genes in the placenta from canonical promoters was lower compared to the adult blood lymphocytes, but the expression of the NUP153 gene from the alternative LINE1 promoter was higher in the placenta. This result has supported the hypothesis that in the placenta, the expression of genes from alternative promoters derived from retroviruses and retrotransposons can be activated due to the hypomethylated epigenetic landscape. This assump tion is also supported by the enrichment of genes that are tissue-specifically expressed in the placenta among all genes which can be transcribed from alternative LINE1 promoters (Criscione et al., 2016a). We have not found significant differences in the level of expression of the YWHAB and NUP153 genes from alterna tive promoters between groups of spontaneous abortions with different karyotypes and the control group of induced abortions. At the same time, the levels of expression of both genes from canonical promoters were higher in the group of spontaneous abortions with monosomy X. However, it has been found that the level of expression of the studied genes changes in individual spontaneous abortions depending on changes in the level of LINE1 methylation. The obtained data clearly demonstrated that the expression level of the NUP153 and YWHAB genes from the canonical and alternative LINE1 promoters correlates with the LINE1 methylation level: the higher the LINE1 methylation level, the lower the expression.
There can be several reasons for the relationship between the level of LINE1 methylation and the expression of the studied genes from both promoters. First, a short transcript from an alternative promoter may be associated with the activation of gene transcription from the canonical promoter. However, this option seems unlikely, because the expression level of the studied genes from canonical promoters against the background of genome hypomethylation in the placenta was lower than in lymphocytes, which are characterized by a LINE1 methylation level of more than 70 % (Rosser, An, 2012). This should be the opposite if this hypothesis is correct.

ЭПИГЕНЕТИКА И РЕГУЛЯЦИЯ АКТИВНОСТИ ГЕНОВ / EPIGENETICS AND GENE REGULATION
Second, the level of methylation of the LINE1 retrotrans poson may reflect the global level of genome methylation and the level of methylation in the canonical promoter of the studied genes. This variant seems to be more likely, but also doesn't remove the issue of reduced expression of the studied genes in the placenta against the background of a hypomethy lated epigenetic landscape compared to adult lymphocytes. The expression of the studied genes is regulated not only by methylation but also by tissue-specific transcription factors. It remains unclear whether the NUP153 and YWHAB gene expression both from the canonical and alternative promoter plays a functional role in the placenta or whether these tran scripts are byproducts of the genome hypomethylation. Po tentially, the impaired NUP153 expression can have a negative impact on the nuclearcytoplasmic transport of proteins and mRNA, and the abnormal YWHAB gene expression can affect the transmission of cell signals.
NUP153 and YWHAB gene products have significant func tional connections with proteins involved in the differentiation of the trophoblast (see Fig. 1). NUP153 interacts with the AGO2, SENP2, C1QBP, and PPARD genes. A list of signifi cant connections is wider for the YWHAB gene -it interacts with the TFEB, CUL7, ZFP36L, MAP2K1, AKT1, CDKN1B, SNAI1, MAPK1, and EGFR genes.
The impaired function of each of these genes has a negative effect on the normal course of embryogenesis. For example, the normal expression of MAPK1 is necessary for the develop ment of nonembryonic ectoderm during placentogenesis. Its absence can lead to embryo death due to abnormal develop ment and hypovascularization of the placenta (Bissonauth et al., 2006). The CUL7 gene is actively expressed in the cell lines of the trophoblast. Protein deficiency of the CUL7 gene is associated with a delay in intrauterine development due to abnormal development of the placenta, which leads to intra uterine hypoxia (Fahlbusch et al., 2012). The deficit can lead to the occurrence of cutaneous or hypodermal hemorrhages, as well as the development of trophoblast with abnormal vas cular structure at later stages of gestation (Arai et al., 2003). CUL7 mutations in the embryo line are associated with the 3M syndrome, which is characterized by pre and postnatal growth retardation (Maksimova et al., 2007;Fu et al., 2010).
The SENP2 gene belongs to the family of ubiquitinlike proteins and is localized in the cell in the nuclear pores and cytoplasm (Talamillo et al., 2020). SENP2 mutations impair cell cycle progression during trophoblast development in mice: deletion of SENP2 impairs the p53/Mdm2 pathway, affect ing trophoblast progenitor cells and their maturation (Chiu et al., 2008). SENP2 influences the normal development of cardiomyocytes during further differentiation. Overexpres sion causes abnormal proliferation of cardiomyocytes with dysregulation of cyclin and cyclindependent kinase inhibi tors, leading to congenital heart anomalies (Kim et al., 2012). On the other hand, deletions also cause defects in myocardial development due to reduced proliferation (Kang et al., 2010).
It is logical to assume that the existing functional relation ships of the NUP153 and YWHAB genes with genes involved in trophoblast differentiation can go both in a negative di rection and in a protective one. Pathological changes in the expression of the NUP153 and YWHAB genes can potentially lead to impaired function of other genes, the formation of a pathological embryo phenotype, or even embryonic death.

Conclusion
We have revealed that the NUP153 and YWHAB genes in the placenta tissues are predominantly expressed from alternative LINE1 promoters located in the intrones. Even though the expression from alternative promoters of LINE1 was higher than with canonical gene promoters for all groups (sponta neous and induced abortions), and there were no significant differences in the level of expression of the YWHAB and NUP153 genes from alternative promotors between groups, we have seen a trend towards the general decrease in expres sion in spontaneous abortions compared to induced abortions. However, it has been found that the level of expression of the studied genes changes in individual spontaneous abortions, depending on changes in the level of genome methylation. The obtained data demonstrate the relationship between the levels of the NUP153 and YWHAB gene expression from canonical and alternative LINE1 promoters with LINE1 methylation levels in extraembryonic tissues of spontaneous abortions.
Thus, an increase in the LINE1 methylation index in the placenta of spontaneous abortions may be associated with a decrease in gene expression not only from alternative but also from canonical promoters. The revealed features of the relationship between the LINE1 methylation level with the NUP153 and YWHAB gene expression levels indicate an exist ing mechanism for selfregulation of normal embryogenesis, disturbance of which can lead to embryo death.